Multiphoton-excited fluorescence of fluorogen-labeled neurotransmitters

Fluorescence detection of fluorogen-labeled neurotransmitters is demonstrated using 100 fs pulses from a titanium-sapphire mode-locked laser to achieve molecular excitation by simultaneous absorption of two and three photons of near-IR radiation. Two-photon excitation spectra are determined for the naphthalene-2,3-dicarboxaldehyde derivative of glycine and the fluorescamine derivative of leucine enkephalin, with the peak excitation cross section (sigma(2)) approximately equal to 1 x 10(-50) cm(4) s/photon for both species. Three-photon-excited fluorescence is demonstrated for o-phthaldialdehyde-labeled glutamate using excitation wavelengths between 965 and 1012 nm. The three-photon excitation cross section (sigma(3)) remains nearly constant in this wavelength range, with an absolute value of similar to 10(-84)-10(-85) cm(6) s(2)/photon(2). Rapid cycling of analytes through the fluorescent excited state and detection that is free from background caused by Rayleigh and Raman scatter combine to make multiphoton-excited fluorescence a highly sensitive approach for detecting trace amounts of neurotransmitters. Measurements of two-photon-excited fluorescence of fluorescamine-labeled bradykinin and analysis of multiphoton-excited background reveal the potential of this method to detect fewer than 1000 neurotransmitter molecules.